WO2017137834A1

WO2017137834A1 - Bacterial growth media

Info

Publication number: WO2017137834A1
Application number: PCT/IB2017/000149
Authority: WO
Inventors: Hanne C. WINTHER-LARSEN; Julia Isabel TANDBERG
Original assignee: University Of Oslo
Priority date: 2016-02-08
Filing date: 2017-02-08
Publication date: 2017-08-17

Abstract

The present invention relates to media for culturing bacteria. In particular, the present invention relates to media for culture of Piscirickettsia salmonis and Fransicella.

Description

BACTERIAL GROWTH MEDIA

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims the priority benefit of U.S. Provisional Patent

Application 62/292,523, filed February 8, 2016, which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to media for culturing bacteria. In particular, the present invention relates to media for culture of Piscirickettsia spp. and Fransicella spp.

BACKGROUND OF THE INVENTION

Fish farming involves raising fish commercially in tanks or enclosures, usually for food. A facility that releases juvenile fish into the wild for recreational fishing or to supplement a species' natural numbers is generally referred to as a fish hatchery. Worldwide, the most important fish species used in fish farming are carp, salmon, tilapia and catfish.

There is an increasing demand for fish and fish protein, which has resulted in widespread overfishing in wild fisheries, China holding 62 percent of the world's fish farming practice. Fish farming offers fish marketers another source. However, farming carnivorous fish, such as salmon, does not always reduce pressure on wild fisheries, since carnivorous farmed fish are usually fed fishmeal and fish oil extracted from wild forage fish. The global returns for fish farming recorded by the FAO in 2008 totaled 33.8 million tons worth about $US 60 billion. In 2005, aquaculture represented 40% of the 157.5 million tons of seafood that was produced, meaning that it has become a critical part of the world's food source even though the industry is still technically in its 'infancy' and didn't really become well known until the 1970s. Because of this rise in aquaculture, there has been a rise in the per capita availability of seafood globally within the last few decades.

A 2008 meta-analysis of available data shows that salmon farming reduces the survival of associated wild salmon populations. This relationship has been shown to hold for Atlantic, steelhead, pink, chum, and coho salmon. The decrease in survival or abundance often exceeds 50 percent.

Diseases of bacterial and viral nature, as well as parasites are the most commonly cited reasons for such decreases. Some bacterial species have been noted to target farmed coho and Atlantic salmon. Because of bacterial disease problems, some aquaculture operators frequently use strong antibiotic drugs to keep the fish alive (but many fish still die prematurely at rates of up to 30 percent). In some cases, these drugs have entered the environment. Additionally, the residual presence of these drugs in human food products has become controversial. Use of antibiotics in food production is thought to increase the prevalence of antibiotic resistance in human diseases. At some facilities, the use of antibiotic drugs in aquaculture has decreased considerably due to vaccinations and other techniques. However, most fish farming operations still use antibiotics, many of which escape into the surrounding environment.

Improved methods for detecting, treating, and preventing infection of fish are needed.

SUMMARY OF THE INVENTION

For example, in some embodiments, the present invention provides a composition, comprising, consisting essentially of, or consisting of at least one of: yeast extract; peptone; (NH₄)₂S0₄; MgS0₄ 7H₂0; K₂HP0₄; NaCl; CaCl₂ 2H₂0; or FeCl_3. In some embodiments, the components are provided in amounts effective to support growth of P. salmonis to an optical density of at least 5, 6, 7, 8, 9, or 10 and up to about 20. In some embodiments, the yeast extract is present at a concentration of approximately 1.0-10.0 g/L, preferably 2.0-8.0 g/L (e.g., 2.0 g/L); the peptone is a peptic digest of meat (e.g., present at a concentration of 1.0- 10.0 g/L, preferably 2.0-8.0 g/L, or approximately 2.0 g/L); the (NH )₂S0₄ is present at a concentration of above 0.8 g/L, e.g., approximately 1.32 g/L; the MgS0₄ 7H₂0 is present at a concentration of 0.1-10.0 g/L (e.g., 0.1 g/L); the K₂HP0₄ is present at a concentration of 1.0- 8.0 g/L, e.g., approximately 6.3 g/L; the NaCl is present at a concentration of 1.0-30.0 g/L, e.g., approximately 9.0 g/L; the CaCl₂ 2H₂0 is present at a concentration of 0.01-0.15 g/L, e.g. approximately 0.08 g/L; and the FeCl₃ is present at a concentration of 10 uM-10 mM, e.g., approximately 2 mM. In some embodiments, the composition further comprises a plurality of amino acids (e.g., serine, alanine, proline, glutamine and glutamic acid). In some embodiments, the amino acids are present at a concentration of approximately 0.01%- 10% (e.g., 0.1%). In some embodiments, the iron is present as FeCl₃, FeS0₄ or FeNH S0₄. In some embodiments, magnesium is present as MgS0₄ 7H₂0 or MgCl₂*6H₂0.

Further embodiments provide a method of culturing bacteria, comprising: incubating the bacteria with the compositions described herein. In some embodiments, the bacterium is a Piscirickettsia species (spp.), preferably P. salmonis. In some embodiments, the bacterium is aFransicella spp., preferably F. noatunensis. In some embodiments, the method further comprises the step of contacting the bacteria with a test compound (e.g., a known or suspected antibiotic). In some embodiments, the method further comprises the step of harvesting the bacteria after incubating. In some embodiments, the present invention provides for the use of the composition described above to culture bacteria such as Piscirickettsia spp., preferably P. salmonis and Fransicella spp., preferably F. noatunensis.

In some embodiments, the present invention provides compositions comprising a liquid bacterial culture of Piscirickettsia spp., preferably P. salmonis and Fransicella spp., preferably F. noatunensis in the culture media described above.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES FIG. 1 shows growth curves of P. salmonis strain LF-89 in Bacto™ Eugon broth (EB) or BMJ (2 g/1 yeast extract, 2 g/1 peptone, 0,1 g/1 MgS04, 6,3 g/1 K2HP04, 9 g/1 NaCl, 0,08 g/1 CaC12) medium supplemented with 2 mM FeC13 (Fe) and a selection of different amino acids.

FIG. 2 shows growth curves of P. salmonis strain LF-89 in Bacto™ Eugon broth (EB) or BMJ (2 g/1 yeast extract, 2 g/1 peptone, 0,1 g/1 MgS04, 6,3 g/1 K2HP04, 9 g/1 NaCl, 0,08 g/1 CaC12) medium supplemented with 2 mM FeC13 (Fe) and a mix of different amino acids.

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below. As used herein, the term "subject" refers to any animal (e.g. , a fish), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g. , with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g. , non-transformed cells), and any other cell population maintained in vitro.

As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g. , an animal or a cell) and to processes or reaction that occur within a natural environment.

The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g. , cancer). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. In some embodiments of the present invention, test compounds include antisense compounds.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention. DETAILED DESCRIPTION OF THE INVENTION

P. salmonis is an intracellular bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry. It is also a pathogen to other fish species (Mauel & Miller, 2002 Vet Microbiol 87:279). Thus, infection by P. salmonis results in both large economic losses and substantial pollution of the environment due to attempts to treat outbreaks of infection with antimicrobial agents.

Until recently, P. salmonis growth could only be performed in fish cell cultures and not standard liquid growth media. Defined culture media have been developed (see, e.g., Yanez et al, Dis Aquat Organ, 2012, 97, 197-205; Henriquez et al., PLOS ONE, 2013, 8(9):e71830; each of which is herein incorporated by reference in its entirety), however culture in these media only results in minimal growth of P. salmonis to an optical density (OD) of 1.8. Likewise, media have been developed which are less defined and whose use is cost prohibitive. For example, EBFC medium comprises BD Bacto Eugon Broth supplemented with 2 mM FeC¾ (Sigma- Aldrich Co., St. Louis, MO, USA) and 1% Casamino Acids (BD). (See e.g., Master thesis of Eva Kathrin Berger, June 2014, University of Oslo). The present invention provides defined growth media for P. salmonis which allow growth to much higher optical densities than most previously described media, and which is more defined and much less expensive to use than EBFC.

The growth media described herein (referred to as BMJ) is a variation of the BM1 media described in Henriquez et al., 2013. The BMI media from Henriquez et al, 2013 comprises: yeast extract (Merck) 2.0 g L-l, peptone from meat (peptic digested, Merck) 2.0 g L-l, (NH₄)₂S0₄ 1.32 g L-l, MgS0₄ 7H₂0 0.1 g L-l, K₂HP0₄ 6.3 g L-l, NaCl 9.0 g L-l, CaCl₂ 2H₂0 0.08 g L-l, FeS0₄ 7H₂0 0.02 g L-l + 5 g L-l glucose. The BMJ media described herein is a modification of the BMI media from Henriques et al, and does not contain glutamate, FeS0₄*7H₂0 and glucose. Accordingly, in some embodiments, the media of the present invention are substantially free of one or more of glutamate, FeS0₄*7H₂0 and glucose.

During development of embodiments of the present invention, the BMJ media was developed by adding 2 mM FeCl₃ (Sigma-Aldrich Co., St. Louis, MO, USA) and 1%

Casamino Acids (BD) (CAAS). This media gave a bacterial growth up to OD = 10. For example, in some embodiments, the present invention provides a composition, comprising at least one of: yeast extract; peptone; (NH₄)₂S0₄; MgS0₄ 7H₂0; K₂HP0₄; NaCl; CaCl₂ 2H₂0; or FeCl_3. In some embodiments, the yeast extract is present at a concentration of

approximately 1.0-10.0 g/L. preferably 2.0-8.0, e.g. approximately 2.0 g/L; the peptone is a peptic digest of meat present at a concentration of 1.0-10.0 g/L, preferably 2.0-8.0 g/L, e.g., present at a concentration of approximately 2.0 g/L; the (NH )₂S0₄ is present at a concentration of above 0.8 g/L, e.g., approximately 1.32 g/L; the MgS0₄ 7H₂0 is present at a concentration of 0.1-10.0 g/L, e.g. approximately 0.1 g/L, ; the K₂HP0₄ is present at a concentration of 1.0-8.0 g/L, e.g. approximately 6.3 g/L; the NaCl is present at a

concentration of 1.0-30.0 g/L, e.g., approximately 9.0 g/L; the CaCl₂ 2H₂0 is present at a concentration of approximately 0.01-0.15 g/L, e.g. approximately 0.08 g/L; and the FeCl₃ is present at a concentration of 10 uM-10 mM, e.g. approximately 2 mM. In some

embodiments, the composition further comprises a plurality of amino acids (e.g., serine, alanine, proline, glutamine and glutamic acid) or 1% Casamino Acids (BD) (CAAS). In some embodiments, the amino acids are present at a concentration of 0.01-10%, e.g. approximately 0.1%.

Sources of iron in the BMJ medium can be FeCl₃, FeS0₄ and FeNH₄S0_4. Magnesium sources can be MgS0₄ 7H₂0 or MgCl₂*6H₂0.

The use of CAAS amino acids in the growth medium EB+Fe+CAAS (EBFC) was previously investigated (Master thesis of Eva Kathrin Berger, June 2014. University of Oslo). In this media it was observed by using mass-spectrometry that the following amino acids were consumed by P. salmonis: serine, alanine, proline, glycine and glutamic acid. Based on this, P. salmonis media was optimized with the BMJ media with the following amino acids: serine, alanine, proline, glycine and glutamic acid. The amino acids are present in

concentrations of 0.01-10%. Thus, in some embodiments, BMJ comprises 0.1 % of serine, alanine, proline, glutamine and glutamic acid.

Growth media based on BMJ, but with only one of these five amino acids, have also been tested. Amino acids were tested one at the time added at a concentration of 0.5 %. This gave growth up to lower ODs than the five amino acid mix, but with the highest growth rate and reaching highest OD with glutamine.

In some embodiments, the present invention provides a growth media comprising: yeast extract (Merck) 2.0 g/L, peptone from meat (peptic digested, Merck) 2.0 g L/l, (NH₄)₂S0₄ 1.32 g L/l, MgS0₄ 7H₂0 0.1 g L/l, K₂HP0₄ 6.3 g L/l, NaCl 9.0 g L/l,

CaCl₂ 2H₂0 0.08 g L/l supplemented with 2 mM FeCl₃ (Sigma-Aldrich Co., St. Louis, MO, USA) and the following amino acids: 0.1 % serine, alanine, proline, glutamine and glutamic acid.

The media described herein is highly beneficial for further studies of P. salmonis as it is a cheaper and more defined media that previously media. In some embodiments, the media described herein finds use in screening of test compounds (e.g., antibiotics) against P.

salmonis and in growing and harvesting bacteria for vaccine development.

Example

P. salmonis LF89 was routinely grown at 20°C on Eugon Chocolate Agar (ECA), containing 30.4 g/L BD Bacto™ Eugon Broth (Becton, Dickinson and Company, Franklin lakes, NJ, USA), 15 g/L Agar Bacteriological (Thermo Fisher Scientific, Hudson, NH, USA) and 5% bovine blood (Hatunalab AB) before being inoculated into either EBFC or BMJ broth supplemented with 2 mM FeCl₃ (Sigma-Aldrich Co., St. Louis, MO, USA) and amino acids (BD) with agitation (100 rpm) for 7-10 days. Bacterial growth was followed over time by measuring OD600 nm in a spectrophotometer. Bacterial stocks used were frozen in autoclaved 10% skimmed milk (BD Difco) or in BD Bacto™ Eugon Broth supplemented with 20% glycerol (Sigma- Aldrich) and stored at - 80°C.

Fig. 1 and Fig 2 show growth curves of P. salmonis strain LF-89 in various growth media. The BMJ media used was yeast extract (Merck) 2.0 g/L, peptone from meat (peptic digested, Merck) 2.0 g L/l, (NH₄)₂S0₄ 1.32 g LA, MgS0₄ 7H₂0 0.1 g L/l, K₂HP0₄ 6.3 g L/l, NaCl 9.0 g L/l, CaCl₂ 2H₂0 0.08 g L/l supplemented with 2 mM FeCl₃ (Sigma- Aldrich Co., St. Louis, MO, USA. The amino acids were present in concentration of 0.1%.

Growth in EB supplemented with 2 mM FeCl₃ and 1% CAAS is compared to growth in BMJ supplemented with 2 mM FeCl₃ and 1% CAAS, or with a selection of different amino acids. In Fig. 1, BMJ is supplied with 2 mM FeCl₃ and one of the amino acids serine, alanine, proline, glutamine and glutamic acid. In Fig. 2, BMJ is supplied with 2 mM FeCl₃ a combination of the amino acids serine, alanine, proline, glutamine and glutamic acid.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in organ storage and transplant, cryobiology, biochemistry, or related fields are intended to be within the scope of the following claims.

Claims

Claims We claim:

1. A composition comprising:

yeast extract; peptone; (NH₄)₂S0₄; MgS0₄ 7H₂0; K₂HP0₄; NaCl; CaCl₂ 2H₂0; and

FeCl₃.

2. The composition of claim 1, wherein said yeast extract is present at a concentration of approximately 1.0-10.0 g/L.

3. The composition of claim 2, wherein said yeast extract is present at a concentration of approximately preferably 2.0-8.0 g/L.

4. The composition of claim 1 or 2, wherein said yeast extract is present at a concentration of approximately 2.0 g/L.

5. The composition of any one of claims 1 to 4, wherein said peptone is peptic digested from meat.

6. The composition of claim 5, wherein said peptone is present at a concentration of approximately 1.0-10.0 g/L.

7. The composition of claim 6, wherein said peptone is present at a concentration of approximately 2.0-8.0 g/L.

8. The composition of claim 7, wherein said peptone is present at a concentration of approximately 2.0 g/L.

9. The composition of any one of claims 1 to 8, wherein said (NH )₂S0₄ is present at a concentration of at least 0.8 g/L.

10. The composition of claim 9, wherein said (NH₄)₂S0₄ is present at a concentration of approximately 1.32 g/L.

11. The composition of any one of claims 1 to 10, wherein said MgSC>4 7H₂0 is present at a concentration of 0.1 -10.0 g/L.

12. The composition claim 11, wherein said MgSCV 7H₂0 is present at a concentration of approximately 0.1 g/L.

13. The composition of any one of claims 1 to 12, wherein said K₂HPO₄ is present at a concentration of 1.0-8.0 g/L.

14. The composition of claim 13, wherein said K₂HPO₄ is present at a concentration of approximately 6.3 g/L.

15. The composition of any one of claims 1 to 14, wherein said NaCl is present at a concentration of 1.0 - 30.0 g/L.

16. The composition of claim 15, wherein said NaCl is present at a concentration of approximately 9.0 g/L.

17. The composition of any one of claims 1 to 16, wherein said CaCl₂ 2H₂0 is present at a concentration of 0.01-0.15 g/L.

18. The composition of claim 17, wherein said CaCl₂ 2H₂0 is present at a concentration of approximately 0.08 g/L.

19. The composition of any one of claims 1 to 8, wherein said FeCl₃ is present at a concentration of 10 uM-10 mM.

20. The composition of claim 19, wherein said FeC¾ is present at a concentration of approximately 2 mM.

21. The composition of any one of claims 1 to 20, wherein said composition further comprises a plurality of amino acids.

22. The composition of claim 21, wherein said amino acids are serine, alanine, proline, glutamine and glutamic acid.

23. The composition of claim 21 or 22, wherein said amino acids are present at a concentration of 0.01-10%.

24. The composition of claim 23, wherein said amino acids are present at a concentration of approximately 0.1%.

25. A method of culturing a bacteria, comprising:

incubating the bacteria with the composition of any one of claims 1 to 24.

26. The method of claim 25, wherein said bacteria is Piscirickettsia salmonis or Fransicella.

27. The method of claim 25 or 26, further comprising the step of contacting said bacteria with a test compound.

28. The method of claim 27, wherein said test compound is a known or suspected antibiotic.

29. The method of claim 27 or 28, further comprising the step of harvesting said Piscirickettsia salmonis or Fransicella after said incubating.

30. Use of the composition of any one of claims 1 to 24 to culture a bacteria.

31. Use of claim 30, wherein said bacteria is Piscirickettsia sp. or Fransicella sp.

32. Use of claim 30 or 31, further comprising the step of contacting said bacteria with a test compound.

33. Use of claim 32, wherein said test compound is a known or suspected antibiotic.

34. Use of claim 30 or 31, further comprising the step of harvesting said Piscirickettsia sp. or Fransicella sp. after said incubating.

35. A composition comprising a liquid culture medium according to any of claims 1 to 24 and a bacteria selected from the group consisting oiPiscirickettsia sp. or Fransicella sp.